The objectives of this proposal are to determine 1) if the nuclear epidermal growth factor receptor (nEGFR) drives resistance to cetuximab in triple-negative breast cancer (TNBC) via its co-transcriptional functions, 2) if targeting nEGFR, by inhibiting its nuclear translocation, can increase the efficacy of cetuximab in TNBC and 3) if we can functionally target nEGFR in TNBC patients. Triple-negative breast cancer (TNBC) is a tumor that lacks estrogen, progesterone and HER2 receptors, leaving minimal therapeutic options. As a result, the prognosis of patients with TNBC remains poor. Recent studies revealed that approximately 50% of TNBC overexpress the oncogene EGFR, which predicts poor patient outcome in TNBC. Unfortunately, clinical trials testing the efficacy of the anti-EGFR monoclonal antibody cetuximab revealed little impact on outcomes in TNBC. This suggests a gap in our knowledge of how EGFR drives TNBC and how to target EGFR effectively in this disease. It is well established that EGFR functions in two distinct signaling compartments: 1) Classical membrane bound signaling and 2) nuclear signaling. Within the nucleus, EGFR performs two functions: 1) as a co-transcription factor enhancing transcription of cancer-promoting genes, and 2) as a tyrosine kinase responsible for phosphorylating substrates that enhance DNA replication and repair. We have published data showing that nuclear EGFR (nEGFR) confers resistance to cetuximab therapy and that the Src Family Kinases (SFKs) are necessary for nuclear translocation of the EGFR. In this proposal we hypothesize that nEGFR contributes to cetuximab resistance via its role as a nuclear co-transcription factor and serves as a functional molecular target in TNBC. To test this hypothesis we have mapped the EGFR C-terminal domain to identify key amino acids necessary for transcriptional activity of the EGFR. This resulted in an EGFR variant that lacks transcriptional activity but retains its signaling capacity that will be used to elucidate nEGFR transcriptional function in cetuximab resistance (Specific Aim 1). This proposal will further assess the therapeutic benefit of targeting both the classical and nEGFR signaling pathways simultaneously by abrogating nuclear translocation via SFK blockade (dasatinib) - followed by subsequent targeting of the classical EGFR signaling pathway with cetuximab in both orthotopic and patient derived xenograft model systems (Specific Aim 2). Finally, we will perform a window trial in nEGFR positive TNBC patients, to determine if we can target nEGFR in humans and redistribute EGFR expression to the plasma membrane where it can be inhibited by cetuximab therapy (Specific Aim 3). Collectively, these studies have potential to re-define how we target EGFR in TNBC and other nEGFR expressing cancers (HNSCC, Lung, bladder), by targeting both compartments of EGFR signaling, and thus turning a tumor that is intrinsically resistant to cetuximab into a tumor that is now sensitive to cetuximab. This investigative strategy can be readily translated to the clinic for treatment of TNBC patients.